Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.
Glucosyltransferases/metabolism* ; Glycoside Hydrolases/metabolism* ; Glycosylation ; Glycosyltransferases/genetics*

The lignan glycosyltransferase UGT236(belonging to the UGT71 B family) from Isatis indigotica can catalyze the production of phloridzin from phloretin in vitro. UGT236 shares high identity with P2'GT from apple. In this study, the recombinant plasmid pET28 a-MBP-UGT236 was transferred into Escherichia coli Rosetta(DE3) cells and induced by isopropyl-β-D-thiogalactoside(IPTG). The purified UGT236 protein was used for enzymatic characterization with phloretin as substrate. The results showed that UGT236 had the optimal reaction temperature of 40 ℃ and the optimal pH 8(Na_2HPO_4-NaH_2PO_4 system). The UGT236 activity was inhibited by Ni~(2+) and Al~(3+), enhanced by Fe~(2+), Co~(2+), and Mn~(2+), and did not affected by Mg~(2+), Ca~(2+), Li~+, Na~+, or K~+. The K_m, K_(cat), and K_(cat)/K_m of phloretin were 61.03 μmol·L~(-1), 0.01 s~(-1), and 157.11 mol~(-1)·s~(-1)·L, and those of UDPG were 183.6 μmol·L~(-1), 0.01 s~(-1), and 51.91 mol~(-1)·s~(-1)·L, respectively. The possible active sites were predicted by homologous modeling and molecular docking. By mutagenisis and catalytic activity detection, three key active sites, Glu391, His15, and Thr141, were identified, while Phe146 was related to product diversity. In summary, we found that the lignan glycosyltransferase UGT236 from I.indigotica could catalyze the reaction of phloretin into phloridzin. Several key amino acid residues were identified by structure prediction, molecular docking, and site-mutagenesis, which provided a basis for studying the specificity and diversity of phloretin glycoside products. This study can provide a reference for artificially producing glycosyltransferase elements with high efficiency and specific catalysis.
Glucosyltransferases/genetics* ; Glycosyltransferases/metabolism* ; Isatis ; Lignans/metabolism* ; Molecular Docking Simulation ; Phloretin/metabolism* ; Phlorhizin/metabolism*

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.
Dendrobium/genetics* ; Plant Growth Regulators ; Glycosyltransferases/metabolism* ; Gene Expression Profiling ; Mycorrhizae ; Phylogeny ; Plant Proteins/metabolism*

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways ; Ginsenosides ; biosynthesis ; Glycosyltransferases ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Synthetic Biology

Glycosyl fluorides are becoming increasingly important molecules for the study on glycosidases. Firstly, glycosyl fluorides act as substrates for glycosidases hydrolysis. Scecondly, the installation of fluorine elsewhere on the carbohydrate ring modifies the properties of the glycosyl fluoride so that the resultant compounds act as mechanism-based inhibitors to label enzymes in the active site, allowing identification of the catalytic nucleophile. Furthermore, glycosyl fluorides also act as donors for transglycosylation by retaining glycolides. Finally, glycosyl fluorides of the wrong anomeric configuration could be used by retaining glycosidase mutants such as glycosynthases and thioglycosynthases to synthesize carbohydrate with high yields(normally 60% to approximately 90%). Fundamental and applied research in biology, glycobiology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo-/poly-saccharides.
Animals ; Enzyme Inhibitors ; chemistry ; Fluorides ; chemistry ; Glucosidases ; metabolism ; Glycoside Hydrolases ; metabolism ; Glycosides ; chemistry ; Glycosyltransferases ; metabolism ; Humans ; Hydrolysis ; Substrate Specificity

Glycosylation, one of the most common and important reactions in biological systems, results in diverse functions and is often found in biologically active small-molecule natural products produced by microorganisms. Furthermore, sugar moieties are generally critical for their activities. Alternating the sugar structures thus provides the potentials for enhancing the biological activities of natural products, which evokes researchers to study the sugar biosynthetic machinery and its application in the modification of sugar moieties with an aim of generating unnaturally glycosylated natural product drugs with better activities. This review will briefly outline current studies on sugar biosynthesis and glycosyltransferase, with a few selected experiments designed to alter natural-product sugar structures.
Anthraquinones ; metabolism ; Biological Products ; chemistry ; metabolism ; Carbohydrates ; biosynthesis ; chemistry ; Erythromycin ; biosynthesis ; Glycosylation ; Glycosyltransferases ; biosynthesis ; Isomerism ; Molecular Structure ; Saccharopolyspora ; metabolism ; Streptomyces ; metabolism ; Synthetic Biology

OBJECTIVETo modify the structure of psoralidin using in vitro enzymatic glycosylation to improve its water solubility and stability.

METHODSA new psoralidin glucoside (1) was obtained by enzymatic glycosylation using a UDP- glycosyltransferase. The chemical structure of compound 1 was elucidated by HR-ESI-MS and nuclear magnetic resonance (NMR) analysis. The high-performance liquid chromatography (HPLC) peaks were integrated and sample solution concentrations were calculated. MTT assay was used to detect the cytotoxicity of the compounds against 3 cancer cell lines in vitro. Results Based on the spectroscopic data, the new psoralidin glucoside was identified as psoralidin-6',7-di-O-β-D- glucopyranoside (1), whose water solubility was 32.6-fold higher than that of the substrate. Analyses of pH and temperature stability demonstrated that compound 1 was more stable than psoralidin at pH 8.8 and at high temperatures. Only psoralidin exhibited a moderate cytotoxicity against 3 human cancer cell lines. Conclusion In vitro enzymatic glycosylation is a powerful approach for structural modification and improving water solubility and stability of compounds.

Antineoplastic Agents ; metabolism ; Benzofurans ; metabolism ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; Coumarins ; metabolism ; Glucosides ; biosynthesis ; Glycosylation ; Glycosyltransferases ; metabolism ; Humans ; Magnetic Resonance Spectroscopy ; Solubility

The present study was designed to perform structural modifications of of neobavaisoflavone (NBIF), using an in vitro enzymatic glycosylation reaction, in order to improve its water-solubility. Two novel glucosides of NBIF were obtained from an enzymatic glycosylation by UDP-glycosyltransferase. The glycosylated products were elucidated by LC-MS, HR-ESI-MS, and NMR analysis. The HPLC peaks were integrated and the concentrations in sample solutions were calculated. The MTT assay was used to detect the cytotoxic activity of compounds in cancer cell lines. Based on the spectroscopic analyses, the two novel glucosides were identified as neobavaisoflavone-4'-O-β-D-glucopyranoside (1) and neobavaisoflavone-4', 7-di-O-β-D-glucopyranoside (2). Additionally, the water-solubilities of compounds 1 and 2 were approximately 175.1- and 4 031.9-fold higher than that of the substrate, respectively. Among the test compounds, only NBIF exhibited weak cytotoxicity against four human cancer cell lines, with IC values ranging from 63.47 to 72.81 µmol·L. These results suggest that in vitro enzymatic glycosylation is a powerful approach to structural modification, improving water-solubility.
Antineoplastic Agents ; metabolism ; pharmacology ; Bacillus ; enzymology ; Cell Line, Tumor ; Colorimetry ; Drug Screening Assays, Antitumor ; Glucosides ; biosynthesis ; chemistry ; Glycosyltransferases ; metabolism ; Humans ; Isoflavones ; biosynthesis ; chemistry ; Molecular Structure ; Solubility

In order to elucidate the expression and molecular genetic background of ABO gene seven samples with ABO discrepancy further identified as bi-specific ABO gene were studied. All these samples were subjected to phenotyping by monoclonal and polyclonal antisera and were then genotyped by direct DNA sequencing and haplotype-sequencing at the exon 6 and 7 of ABO gene. As a result, six ABO dual-specific alleles were identified in Chinese population. An antigen expressed by these B (A) or Cis-AB individuals varied from very low level to the normal level, compared with common A blood group samples. In conclusion, molecular genetic backgrounds of two pairs out of four samples in all samples were the same, however, the ABO expression showed diverse.
ABO Blood-Group System ; genetics ; Asian Continental Ancestry Group ; genetics ; DNA Mutational Analysis ; Erythrocytes ; cytology ; enzymology ; metabolism ; Exons ; genetics ; Glycosyltransferases ; chemistry ; genetics ; Humans

As more bioactivities of oligosaccharides have been elucidated, researches on biosynthesis of oligosaccharides have drawn more concerns in Glycobiology. A lot of enzymatic methods for the synthesis of oligosacchrides have been developed employing recombinant E. coli expressed glycosyltranferase or synthase of nucleotide-sugar. This review focuses on the recent progress in the production of oligosaccharides using bacteria especially by genetically engineered bacteria. The key point concering the oligosaccharides biosynthesis in recombinant E. coli, such as enzyme expression, NDP-sugar provision and biosynthesis pathway, was discussed.
Bacteria ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Carbohydrate Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Engineering ; methods ; trends ; Glycosyltransferases ; genetics ; metabolism ; Industrial Microbiology ; methods ; trends ; Molecular Sequence Data ; Oligosaccharides ; biosynthesis